Method for preparing hispidulin and its derivatives

ABSTRACT

Provided is a method for preparing hispidulin or a derivative thereof. The method includes selective protection of trihydroxybenzaldehyde, followed by regioselective iodination, selective protection, Stille coupling, Baeyer-Villiger oxidation and basic hydrolysis to obtain a protected intermediate compound. Then, alkylation, Claisen-Schmidt condensation, cyclization and deprotection of the protected intermediate compound are performed to obtain hispidulin or the derivative thereof. The present disclosure provides an efficient method for total synthesis of hispidulin or the derivative thereof with concise reaction steps and high yield.

BACKGROUND 1. Technical Field

The present disclosure relates to a method for preparing hispidulin or aderivative thereof, and, for example, to a method for total synthesis ofhispidulin and d-hispidulin (deuterium-labeled or d-labeled hispidulin).

2. Description of Associated Art

Hispidulin (6-methoxy-4′,5,7-trihydroxyflavone) is a naturally occurringflavone belonging to a group of polyphenolic compounds named flavonoids,which occur ubiquitously in plants. Flavonoids are found to have manybiological properties, such as antioxidative [1], anti-inflammatory [2],antimicrobial [3], anticonvulsant [4], antidepressant [5] and anticancer[6] activities.

Hispidulin is known to have various central nervous system activities.In particular, it was discovered as the main active ingredient from theleaf extract of Clerodendrum inerme (L.) Gaertn (CI) [7] thatsignificantly attenuated methamphetamine-induced hyperlocomotion (MIH)as a mouse model of motor tic, while not affecting the spontaneouslocomotor activity or performance in mice even in amounts up to 100mg/kg [8]. Further investigations showed that hispidulin formed strongbonds with GABA_(A) receptors (IC₅₀=0.73 μM to 1.78 μM) and inhibitedcatecholamine-O-methyl-transferase (COMT) (IC₅₀=1.32 μM) [9]. By actingas a positive allosteric modulator (PAM), hispidulin enhanced cerebellarα₆GABA_(A) receptor activity. It was noted that hispidulin had no hit onhuman ether-á-go-go-related gene (hERG) channels, an undesirable targetin drug development [9], implying that hispidulin is a potentialcandidate for drug development.

Because of its interesting biological activity, there are severalsynthetic approaches developed to synthesize hispidulin [10-15]. Forexample, Shen and coworkers developed a strategy for semisynthesis ofhispidulin in seven reaction steps by using a naturally occurringscutellarin (Scheme 1) [14]. Although this method is concise and has anoverall yield as high as 10.7%, the researchers showed that, upon thelarge-scale synthesis of Formula 1, the protection of the catecholmoiety of scutellarein using dichlorodiphenylmethane at 175° C. failed[12, 15]. While this problem was later solved through a seven-stepsynthesis route developed by Lin and coworkers, however the overallyield was reduced to 7.1% (Scheme 1) [12]. Zhang and coworkers thendeveloped a scheme that only required four reaction steps (Scheme 1),but the nonselective methoxymethyl (MOM) protection of scutellareincaused the decrease in the overall yield of the synthesis of hispidulin(6.3%) [15]. Despite the satisfactory overall yield of these strategies,the tedious purification procedure required to isolate scutellarin fromplants limits their use for large-scale preparation of hispidulin.

Kavvadias and coworkers developed a method synthesizing hispidulin from2,4,6-trihydroxyacetophenone in nine reaction steps (Formula 6 in Scheme2 below). However, the overall yield of this method is very limited(1.1%) [10].

Scheme 3 below shows yet another approach aimed for a feasible andreproducible synthesis of hispidulin [13]. However, this method onlyslightly improved the overall yield due to the low yield ofFriedel-Crafts acetylation of Formula 8 as well as unsatisfactoryregioselective MOM protection of Formula 9.

Accordingly, the previously proposed synthetic approaches to synthesizehispidulin have proven unsatisfactory due to their low feasibility andpoor overall yields. There remains a need for an efficient andhigh-yield method to synthesize hispidulin.

SUMMARY

In view of the foregoing, the present disclosure provides an improvedprocess for preparation of hispidulin or a derivative thereof. A uniqueand efficient strategy for synthesizing hispidulin or a derivativethereof is provided by the present disclosure.

In one embodiment, a new entity of hispidulin derivatives, i.e., adeuterium-labeled hispidulin, is also described in the presentdisclosure. In one embodiment, the deuterium-labeled hispidulin is ofthe formula

wherein R is CD₃.

Another embodiment of the present disclosure provides a method forpreparing hispidulin or a derivative thereof fromtrihydroxybenzaldehyde, the method comprising providing an intermediatecompound of following Formula (IV):

wherein PG₁, PG₂ and PG₃ are each independently a hydroxyl protectinggroup.

In one embodiment, the hydroxyl protecting group is selected from thegroup consisting of methoxymethyl (MOM), 2-methoxyethoxymethyl (MEM),ethoxymethyl (EOM), t-butoxymethyl, benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), allyloxymethoxy, tetrahydropyranyl(THP), methylthiomethyl (MTM), tri-1-propylsilyloxymethyl (TOM),(phenyldimethylsilyl)methoxymethyl (SMOM), acetyl, pivaloyl (Piv),benzoate, methyl, ethyl, benzyl (Bn), p-methoxybenzyl (PMB),triphenylmethyl (Tr), methoxytrityl (MMT), dimethoxytrityl (DMT),trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), triisopropylsilyl(TIPS), tri(trimethylsilyl)silyl (TTMSS), and t-butyldiphenylsilyl(TBDPS).

In another embodiment, the trihydroxybenzaldehyde is2,4,6-trihydroxybenzaldehyde.

In yet another embodiment, the hispidulin or the derivative thereofprepared by the method of the present disclosure is of the formula

wherein R is hydrogen, an optionally substituted alkyl or an optionallysubstituted cycloalkyl. In an embodiment, the optionally substitutedalkyl is CH₃, C₂H₅, or C₃H₇. In another embodiment, the optionallysubstituted cycloalkyl is C₆H₅CH₂.

In still yet another embodiment, the hispidulin derivative prepared bythe method of the present disclosure is deuterium-labeled. In anembodiment, the deuterium-labeled hispidulin derivative has the formula

wherein R is CD₃.

In one embodiment, the intermediate compound of Formula (IV) is obtainedby Baeyer-Villiger oxidation and basic hydrolysis of a compoundrepresented by following Formula (V):

wherein PG₁, PG₂ and PG₃ are as defined above.

In one embodiment, Stille coupling is conducted to obtain the compoundrepresented by Formula (V). In one embodiment, the Stille coupling isconducted with a palladium catalysts, such as Pd(PPh₃)₄, PdCl₂(PPh₃)₂,Pd(dppf)Cl₂, PdCl₂, Pd(OAc)₂, Pd(dba)₂, PdCl₂(MeCN)₂, BnPdCl(PPh₃)₂ andC₄H₆Br₂N₂Pd, coupled with an organic solvent, such as toluene, dioxane,tetrahydrofuran, acetonitrile, chloroform, dichloromethane,chlorobenzene, dimethylacetamide, methylpyrrolidone, dimethyl sulfoxideand hexamethylphosphoramide, or water. In a further embodiment, theStille coupling is conducted with a yield of at least 60%, 70% or 80%.In still yet another embodiment, the Stille coupling is conducted with areaction time of not more than 11 hours, 12 hours, 13 hours, 15 hours or18 hours.

In another embodiment, the trihydroxybenzaldehyde reacts with at leastone protecting group to obtain a compound represented by followingFormula (VII)

wherein PG₂ and PG₃ are as defined above, wherein the PG₂ and the PG₃may be the same or different.

In yet another embodiment, the compound represented by Formula (VII)undergoes regioselective iodination and reacts with an additionalprotecting group to obtain a compound represented by following Formula(VI):

wherein PG₁, PG₂ and PG₃ are as defined above.

In one embodiment, the present disclosure provides a method forsynthesizing the hispidulin or the derivative thereof with a yield of atleast 12%, 15%, 18%, 20%, 25% or 26%.

In another embodiment, the compound represented by Formula (IV)undergoes alkylation and Claisen-Schmidt condensation, followed bydeprotection to obtain a compound represented by following Formula (IA):

wherein PG₁ and PG₄ are each independently a hydroxyl protecting groupas defined above, or PG₄ is independently H, and R is hydrogen, anoptionally substituted alkyl or an optionally substituted cycloalkyl.

In a further embodiment, cyclization of the compound represented byFormula (IA) is conducted in the presence of a catalyst to obtain acompound represented by following Formula (IB):

and deprotection of the compound represented by Formula (IB) isconducted to obtain the hispidulin or the derivative thereof, whereinPG₁, PG₄ and R are as defined above. In yet another embodiment, thecatalyst in the cyclization is catalytic I₂, potassium iodide, ammoniumiodide, tetra-(n-butyl)ammonium iodide, selenium (IV) oxide, dihydrogenperoxide, cerium (IV) sulfate tetrahydrate,2,3-dicyano-5,6-dichloro-p-benzoquinone or bis(acetoxy)iodobenzene. Inanother embodiment, the deprotection of the compound represented byFormula (IB) is debenzylation in a reaction with BCl₃, hydrogen, 10 wt.% palladium on activated carbon, titanium tetrachloride, borontribromide, acetic acid or methanesulfonic acid. In still yet anotherembodiment, the deprotection of the compound represented by Formula (IB)is debenzylation in a reaction with BCl₃ at −80° C.

In one embodiment of the present disclosure, the method of synthesizinghispidulin or a derivative thereof comprises:

reacting 2,4,6-trihydroxybenzaldehyde with at least one protecting groupto obtain a compound represented by following Formula (VII):

conducting regioselective iodination of the compound represented byFormula (VII), and then reacting with an additional protecting group toobtain a compound represented by following Formula (VI):

conducting Stille coupling of the compound represented by Formula (VI)to obtain a compound represented by following Formula (V):

conducting Baeyer-Villiger oxidation and basic hydrolysis of thecompound represented by Formula (V) to obtain an intermediate compoundrepresented by Formula (IV):

alkylating the intermediate compound represented by Formula (IV) toobtain a compound represented by following Formula (IIA):

conducting Claisen-Schmidt condensation and deprotection of the compoundrepresented by Formula (IIA) to obtain a compound represented byfollowing Formula (IA):

conducting cyclization of the compound represented by Formula (IA) inthe presence of a catalyst to obtain a compound represented by followingFormula (IB):

and

deprotecting the compound represented by Formula (IB) to obtain thehispidulin or the derivative thereof,

wherein PG₁, PG₂ and PG₃ each independently represent one of thefollowing structures:

R is hydrogen, an optionally substituted alkyl or an optionallysubstituted cycloalkyl, and PG₄ is H or the same as defined in PG₁, PG₂and PG₃.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the retrosynthetic analysis for hispidulin synthesis.

FIG. 2 shows the synthesis scheme for hispidulin and d-hispidulin.Reagents and conditions: (a) methoxymethyl chloride (MOMCl),N,N-diisopropylethylamine (DIPEA), CH₂Cl₂, 0° C., 80%; (b) (1) CF₃COOAg,I₂, CH₂Cl₂, 0° C.; (2) K₂CO₃, benzyl bromide (BnBr), dimethylformamide(DMF), 0° C., 89%; (c) tributyl(1-ethoxyvinyl)tin, PdCl₂(PPh₃)₂,toluene, 100° C., 83%; (d) (1) meta-chloroperoxybenzoic acid (MCPBA),CH₂Cl₂, 0° C.; (2) 10% NaOH_((aq)), MeOH, 68%; (e) 23a: CH₃I, K₂CO₃,acetone, 56° C., 92%; 23b: CD₃I, K₂CO₃, acetone, 56° C., 93%; (f) (1)BnOPhCHO, KOH, EtOH, H₂O, 0° C.; (2) c-HCl, MeOH, tetrahydrofuran (THF),0° C.; 24a: 92%, 24b: 81%; (g) 12, dimethyl sulfoxide (DMSO), 120° C.,25a: 93%, 25b: 79%; (h) 1M BCl3 in hexane; CH2Cl2, −80° C., hispidulin:85%, d-hispidulin: 80%.

FIG. 3 shows the rotating frame nuclear Overhauser effect spectroscopy(ROESY) correlations of Formula 21.

FIG. 4 shows the ROESY correlations and heteronuclear multiple bondcorrelations (HMBCs) of hispidulin.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following specific examples are used to exemplify the presentdisclosure. A person of ordinary skill in the art can conceive the otheraspects of the present disclosure, based on the disclosure of thespecification. The present disclosure can also be implemented or appliedas described in different specific examples. It is possible to modify oralter the above examples for carrying out this disclosure withoutcontravening its spirit and scope for different aspects andapplications.

Strategies for synthesizing hispidulin can be classified assemisynthesis and total synthesis strategies. The starting material usedin most semisynthetic methods is scutellarin, which is a naturalproduct. Table 1 below shows that the semisynthesis routes had fewerreaction steps compared to the total synthesis methods; however, theyneed tedious isolation procedures for scutellarin, which limits thescale for further chemical modification. Furthermore, their overallyields are only 6.3% to 10.7%.

TABLE 1 Comparison of hispidulin synthesis methods. Reaction OverallYield Synthesis Method by Synthesis Route Steps (%) Shen and coworkersSemisynthesis Seven 10.7 Lin and coworkers Semisynthesis Seven 7.1 Zhangand coworkers Semisynthesis Four 6.3 Kavvadias and coworkers Totalsynthesis Nine 1.1 Chao and coworkers Total synthesis Ten 1.6 Thisdisclosure Total synthesis Eight 26.9

For total synthesis, Kavvadias and coworkers developed a nine-stepsynthesis approach. The starting material used in this method iscommercially available 2,4,6-trihydroxyacetophenone. Although thismethod solves the issue of the source for starting material, itsdrawback is a low overall yield [10]. Chao and coworkers also developeda feasible route of the hispidulin synthesis that has an overall yieldcomparable to that of the method developed by Kavvadias and coworkers[13]. The present disclosure provides a method for total synthesis ofhispidulin with eight reaction steps. The present disclosure provides asynthesis method with more concise reaction steps, which has the highestoverall yield of all currently known approaches to synthesizehispidulin.

In the present disclosure, retrosynthetic analysis for synthesizinghispidulin is carried out and shown in FIG. 1. The retrosyntheticanalysis showed that hispidulin can be produced from Formula (I) viadebenzylation and oxidative cyclization. Formula (I) is derived fromFormula (II) and commercially available Formula (III), which are used toconduct Claisen-Schmidt condensation and deprotection of protectinggroups (PGs). Formula (II) in turn can be prepared from Formula (IV) viamethylation. Formula (IV) is considered to be the intermediate thatpossesses the same or different protecting groups as well as acetyl andhydroxy moieties. Selective Bayer-Villiger reaction of Formula (V)provided Formula (IV). Formula (V) is prepared from Formula (VI) viaStille coupling. Substitution with the protecting groups andregioselective iodination of Formula (VII) gives Formula (VI). Startingfrom commercially available Formula 7, Formula (VII) is synthesized viaselective protection.

Based on the retrosynthetic analysis, the present disclosure provides anovel synthesis method for hispidulin. In one embodiment of the presentdisclosure, as the scheme shown in FIG. 2, a method for preparation ofhispidulin comprises reacting 2,4,6-trihydroxybenzaldehyde (Formula 7)with MOMCl to obtain the bis(methoxymethoxy)-protected compoundrepresented by Formula 20; conducting regioselective iodination of thecompound of Formula 20 and then reacting with BnBr to obtain thecompound of Formula 21; conducting Stille coupling of the compound ofFormula 21 to obtain the compound of Formula 22; conductingBaeyer-Villiger oxidation and basic hydrolysis of the compound ofFormula 22 to obtain the compound of Formula 14; methylating thecompound of Formula 14 by using CH₃I or CD₃I to obtain the compound ofFormula 23 (wherein the compound of Formula 23a contains R as being CH₃,and the compound of Formula 23b contains R as being CD₃); conductingClaisen-Schmidt condensation of Formula 23 by 4-(benzyloxy)benzaldehydeprior to MOM deprotection to obtain chalcone represented by Formula 24(wherein chalcone represented by Formula 24a contains R as being CH₃,and chalcone represented by Formula 24b contains R as being CD₃);cyclizing chalcone represented by Formula 24 in the presence ofcatalytic I₂ to obtain flavone represented by Formula 25 (whereinflavone represented by Formula 25a contains R as being CH₃, and flavonerepresented by Formula 25b contains R as being CD₃); conductingdebenzylation of flavone represented by Formula 25 in a reaction usingBCl₃ at −80° C. to obtain hispidulin or d-hispidulin.

The present disclosure further provides a synthesis method fordeuterium-labeled (d-labeled) hispidulin. Deuterium is a stable isotopeof hydrogen. Because deuterium has a stronger chemical bond with carbonthan hydrogen, deuterium-labeled compounds can affect the absorption,distribution, metabolism and toxicology of their counterpart compounds[16, 17].

In the present disclosure, C₆—OCH₃ in hispidulin is replaced withC₆-OCD₃. The synthesis of d-hispidulin according to the presentdisclosure is described in FIG. 2, wherein Formula 14 is methylated byusing CD₃I to obtain Formula 23b; Claisen-Schmidt condensation ofFormula 23b is conducted with 4-(benzyloxy)benzaldehyde prior to MOMdeprotection to obtain chalcone 24b; chalcone 24b is cyclized in thepresence of catalytic I₂ to obtain flavone 25b; and debenzylation offlavone 25b is conducted in a reaction using BCl₃ at −80° C. to obtaind-hispidulin.

The present disclosure therefore also provides a new hispidulinderivative entity, i.e., a deuterium-labeled (d-labeled) hispidulin.

As used herein, the terms “hydrogen or hydroxyl protecting group” referto protecting groups that protect hydrogen or hydroxy groups. It is tobe understood that such protecting groups are conventional and routinelyselected to allow a synthetic or chemical transformation to be performedin a manner that the hydroxy group does not interfere with or is notchanged by the synthetic or chemical transformation performed.Illustrative, but not exclusive, examples of such protecting groups maybe found in Greene & Wuts “Protective Groups in Organic Synthesis,” 2ndEd., John Wiley & Sons, New York, 1991; the disclosure of which isincorporated herein by reference. Further illustrative of suchprotecting groups are those particularly suitable for protecting phenolsand catechols, and the analogs and derivatives thereof.

As used herein, the term “alkyl” refers to a saturated monovalent chainof carbon atoms, which may be optionally branched. It is understood thatin embodiments that include alkyl, illustrative variations of thoseembodiments include lower alkyl, such as C₁-C₆ alkyl, C₁-C₄ alkyl,methyl, ethyl, propyl, 3-methylpentyl, and the like.

As used herein “cycloalkyl” is intended to indicate a saturatedcycloalkane hydrocarbon radical, comprising 3-6 carbon atoms, e.g.,cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The new hispidulin synthesis method disclosed in the present disclosureis more feasible compared to all methods previously reported. Inparticular, the present disclosure has the highest overall yield, andsynthesizes C₆—OCH₃-containing hispidulin derivatives efficiently. Thesynthesis method disclosed in the present disclosure can also be used tosynthesize d-labeled hispidulin. The present disclosure also provides amethod to produce 6-OMe-containing hispidulin derivatives as newchemical entities.

The following are specific examples further demonstrating theimplementation of the current disclosure, but not to limit the scope ofthe current disclosure.

EXAMPLES

General materials and methods used in the following examples aredescribed herein. The NMR spectra (¹H- and ¹³C-NMR, ROESY, HSQC andHMBC) were obtained with a Bruker AV500 using standard pulse programs.The MS data were recorded with a Finnigan Mat TSQ-7000 mass spectrometer(HR-ESI-MS) (Thermo, Ringoes, N.J., USA). The HPLC was performed on aCis column (150 mm×4.6 mm, Ascentis) by using an L-2130 pump (Hitachi,Ibaraki, Japan) and a UV/vis L-2420 detector (Hitachi, Ibaraki, Japan).The column chromatography was performed on silica gel (70-230 mesh,Merck, Darmstadt, Germany). All TLC analyses were performed on silicagel plates (KG60-F254, Merck, Darmstadt, Germany). Reagents andmaterials were used without further purification, and chemicals werepurchased from ACROS (Geel, Belgium). Dry dichloromethane was distilledfrom CaH₂ under nitrogen atmosphere. MOMCl was acquired from TCI (Tokyo,Japan), and 2,4,6-trihydroxybenzaldehyde was purchased from Alfa Aesar(Heydham, UK).

Example 1: Preparation of 2-Hydroxy-4,6-bis(methoxymethoxy)benzaldehyde(Formula 20)

To a solution of Formula 7 (10 g, 64.9 mmol) in CH₂Cl₂ (200 mL) wasadded DIPEA (28.3 mL, 162.2 mmol). The resulting mixture was stirred for10 min in an ice-bath under N₂. MOMCl (10.8 mL, 142.7 mmol) was addeddropwise to the reaction mixture by an addition funnel. The reactionmixture was warmed to room temperature (rt) and stirred for 3 h. Themixture was concentrated in vacuo. The residue was diluted with EtOAc(100 mL) and washed with distilled H₂O (3×80 mL). The organic layer wasdried over Na₂SO₄, filtered and removed in vacuo. The residue waspurified by silica gel chromatography (EtOAc:n-hexane=1:9) to obtainFormula 20 (12.3 g, 80%), light-yellow microcrystalline powder; ¹H-NMR(CDCl₃, 300 MHz) δ 12.29 (1H, s), 10.16 (1H, s), 6.25 (1H, d, J=2.1 Hz),6.23 (1H, d, J=2.1 Hz), 5.24 (2H, s), 5.18 (2H, s), 3.51 (3H, s), 3.47(3H, s); ¹³C-NMR (CDCl₃, 125 MHz) δ 192.1, 165.6, 165.5, 161.2, 106.9,96.6, 94.6, 94.1, 94.0, 56.6, 56.5; HR-ESI-MS m/z 243.0858 [M+H]⁺(calcd. for C₁₁H₁₅O₆, 243.0863).

Example 2: Preparation of 2-Benzyloxy-3-iodo-4,6-bis(methoxymethoxy)benzaldehyde (Formula 21)

To a mixture of Formula 20 (7.2 g, 29.6 mmol) and CF₃CO₂Ag (7.8 g, 35.5mmol) in CH₂Cl₂ (150 mL) at 0° C. was added I₂ (8.3 g, 32.5 mmol) inCH₂Cl₂ (200 mL) dropwise by an addition funnel over 2 h. The reactionmixture was warmed to rt and stirred for 3 h. Then, saturatedNa₂S₂O_(3(aq)) (50 mL) was added, and the mixture was washed withdistilled H₂O (3×100 mL). The organic layer was dried over Na₂SO₄,filtered and removed in vacuo. The residue was dissolved in DMF (100mL), and then K₂CO₃ (7.2 g, 51.8 mmol) was added. Benzyl bromide (460μL, 3.9 mmol) was added dropwise to the mixture by an addition funnel at0° C. The resulting solution was warmed to rt and stirred for 3 h. Themixture was diluted with EtOAc (300 mL) and washed with distilled H₂O(3×100 mL). The organic layer was dried over Na₂SO₄, filtered andremoved in vacuo. The residue was purified by silica gel chromatography(EtOAc:n-hexane=1:5) to obtain Formula 21 (12.1 g, 89%), whitemicrocrystalline powder; ¹H-NMR (CDCl₃, 300 MHz) δ 10.30 (1H, s), 7.66(2H, dd, J=1.8, 8.1 Hz), 7.45-7.36 (3H, m), 6.84 (1H, s), 5.32 (2H, s),5.29 (2H, s), 4.99 (2H, s), 3.54 (3H, s), 3.52 (3H, s); ¹³C-NMR (CDCl₃,125 MHz) δ 187.2, 161.9, 161.8, 161.6, 136.1, 129.0, 128.5, 128.4,115.5, 98.2, 95.2, 94.9, 78.5, 56.7; HR-ESI-MS m/z 459.0294 [M+H]⁺(calcd. for C₁₈H₂₀O₆I, 459.0299). The chemical structure of Formula 21was confirmed by rotating frame nuclear Overhauser effect spectroscopy(ROESY) spectrum. FIG. 3 shows the correlation of H-5 (δH 6.84) to H-2′″(δH 3.52), H-2″″ (δH 3.54), H-1′″ (δH 5.29) and H-1″″ (δH 5.32); andH-1′ (δH 10.30) to H-2″″ (δH 3.54), H-1″ (δH 4.99) and H-1″″ (δH 5.32).

Example 3: Preparation of 3-Acetyl-2-benzyloxy-4,6-bis(methoxymethoxy)benzaldehyde (Formula 22)

Table 2 below shows how tributyl(1-ethoxyvinyl)tin was used to optimizeStille coupling.

TABLE 2 Optimization of reaction condition for Stille coupling ofFormula 21. Entry Catalyst Solvent Yield (%) Reaction Time (h) 1Pd(PPh₃)₄ Dioxane 68 30 2 Pd(PPh₃)₄ Toluene 70 24 3 CCPdCl₂(PPh₃)₂Dioxane 73 20 4 PdCl₂(PPh₃)₂ Toluene 83 10 5 Pd(dppf)Cl₂ Dioxane 34 78 6Pd(dppf)Cl₂ Toluene 53 43

First, catalyst Pd(PPh₃)₄ was used in dioxane at 100° C. The reactionhad a satisfactory yield (68%), but the reaction time was up to 30 h.Replacement of the solvent by toluene led to the decrease of thereaction time to 24 h. Further experiments using palladium catalystssuch as PdCl₂(PPh₃)₂ and Pd(dppf)Cl₂ in dioxane or toluene showed thatPdCl₂(PPh₃)₂ significantly improved the yield and decreased the reactiontime. For example, PdCl₂(PPh₃)₂ coupled with toluene not only gave thehighest yield, but also had the lowest reaction time.

To a mixture of Formula 21 (5 g, 10.9 mmol) and PdCl₂(PPh₃)₂ (766 mg,1.1 mmol) in toluene (200 mL) was added tributyl(1-ethoxyvinyl)tin (5.5mL, 16.4 mmol). The resulting solution was heated to 100° C. and stirredfor 12 h. After cooling to rt, the reaction mixture was acidified with 1M HCl (50 mL) and stirred for 30 min. The mixture was diluted with EtOAc(200 mL) and washed with distilled H₂O (3×100 mL). The organic layer wasdried over Na₂SO₄, filtered and concentrated in vacuo. The residue waspurified by silica gel chromatography (EtOAc:n-hexane=1:4) to obtainFormula 22 (3.5 g, 83%), yellow microcrystalline powder; ¹H-NMR (CDCl₃,300 MHz) δ 10.37 (1H, s), 7.48 (2H, dd, J=1.8, 7.8 Hz), 7.41-7.33 (3H,m), 6.80 (1H, s), 5.29 (2H, s), 5.24 (2H, s), 4.96 (2H, s), 3.53 (3H,s), 3.49 (3H, s), 2.43 (3H, s); ¹³C-NMR (CDCl₃, 125 MHz) δ 200.9, 187.3,162.1, 159.3, 158.4, 136.1, 129.0, 128.8, 128.6, 128.5, 128.4, 121.8,114.3, 97.5, 95.0, 94.5, 79.1, 56.8, 32.5; HR-ESI-MS m/z 375.1432 [M+H]⁺(calcd. for C₂₀H₂₃O₇, 375.1438).

Example 4: Preparation of 2-Benzyloxy-3-hydroxy-4,6-bis(methoxymethoxy)acetophenone (Formula 14)

To a solution of 70% MCPBA (6.8 g, 27.6 mmol) in dry CH₂Cl₂ (100 mL) at0° C. was added Formula 22 (3.4 g, 9.2 mmol) in CH₂Cl₂ (100 mL) dropwiseby an addition funnel over 1 h. The resulting solution was warmed to rtand stirred for 8 h. Then, saturated Na₂S₂O_(3(aq)) (30 mL) was added,and the reaction mixture was washed with distilled H₂O (3×100 mL). Theorganic layer was dried over Na₂SO₄, filtered and concentrated in vacuo.The residue was dissolved in MeOH (80 mL) and 10% NaOH_((aq)) (60 mL)was added. The resulting solution was stirred at rt for 1.5 h. Themixture was diluted with EtOAc (250 mL) and washed with distilled H₂O(3×100 mL). The organic layer was dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was purified by silica gelchromatography (EtOAc:n-hexane=1:4) to obtain Formula 14 (2.3 g, 68%),light-yellow oil; ¹H-NMR (CDCl₃, 300 MHz) δ 7.44 (2H, dd, J=1.8, 8.1Hz), 7.40-7.32 (3H, m), 6.76 (1H, s), 5.61 (1H, s), 5.21 (2H, s), 5.08,(2H, s), 5.06 (2H, s), 3.53 (3H, s), 3.46 (3H, s), 2.45 (3H, s); ¹³C-NMR(CDCl₃, 125 MHz) δ 201.3, 146.6, 146.2, 143.2, 136.9, 134.9, 128.5,128.3, 122.1, 100.4, 96.0, 95.8, 76.3, 56.6, 56.3, 32.6; HR-ESI-MS m/z363.1431 [M+H]⁺ (calcd. for C₁₉H₂₃O₇, 363.1438).

Example 5: Preparation of 2-Benzyloxy-3-methoxy-4,6-bis(methoxymethoxy)acetophenone (Formula 23a)

To a mixture of Formula 14 (587 mg, 1.6 mmol) and K₂CO₃ (1.1 g, 8.1mmol) in acetone (20 mL) was added CH₃I (0.5 mL, 8.1 mmol). Theresulting solution was heated to 56° C. and stirred for 5 h. Thereaction mixture was concentrated in vacuo, diluted with EtOAc (100 mL)and washed with distilled H₂O (3×50 mL). The organic layer was driedover Na₂SO₄, filtered and concentrated in vacuo. The residue waspurified by silica gel chromatography (EtOAc:n-hexane=1:5) to obtainFormula 23a (558 mg, 92%), white microcrystalline powder; ¹H-NMR (CDCl₃,300 MHz) δ 7.42 (2H, dd, J=2.0, 8.3 Hz), 7.40-7.32 (3H, m), 6.76 (1H,s), 5.23 (2H, s), 5.11 (2H, s), 5.07 (2H, s), 3.85 (3H, s), 3.53 (3H,s), 3.46 (3H, s), 2.38 (3H, s); ¹³C-NMR (CDCl₃, 125 MHz) δ 200.6, 151.8,149.4, 149.2, 137.9, 136.7, 128.1, 128.0, 127.7, 121.4, 99.6, 95.0,94.9, 76.0, 60.8, 56.0, 55.9, 32.1; HR-ESI-MS m/z 377.1587 [M+H]⁺(calcd. for C₂₀H₂₅O₇, 377.1595).

Example 6: Preparation of2-Benzyloxy-3-[2H3]-methoxy-4,6-bis(methoxymethoxy) acetophenone(Formula 23b)

Following the procedure as described for Formula 23a, the reaction ofFormula 14 (1.8 g, 4.8 mmol), K₂CO₃ (3.3 g, 24.2 mmol), and CD₃I (1.5mL, 24.2 mmol) in acetone (40 mL) gave Formula 23b (1.7 g, 93%), whitemicrocrystalline powder; ¹H-NMR (CDCl₃, 300 MHz) δ 7.42 (2H, dd, J=2.1,8.4 Hz), 7.39-7.31 (3H, m), 6.76 (1H, s), 5.23 (2H, s), 5.11 (2H, s),5.07 (2H, s), 3.53 (3H, s), 3.46 (3H, s), 2.38 (3H, s); ¹³C-NMR (CDCl₃,125 MHz) δ 200.6, 151.8, 149.4, 149.2, 137.8, 136.7, 128.1, 128.0,127.7, 121.4, 99.6, 95.0, 94.9, 76.0, 56.0, 55.9, 32.1; HR-ESI-MS m/z380.1775 [M+H]⁺ (calcd. for C₂₀H₂₂D₃O₇, 380.1783).

Example 7: Preparation of(E)-1-(2-Benzyloxy-4,6-dihydroxy-3-methoxyphenyl)-3-(4-benzyloxyphenyl)prop-2-en-1-one(Formula 24a)

To a solution of Formula 23a (545 mg, 1.45 mmol) and4-benzyloxybenzaldyde (615 mg, 2.9 mmol) in EtOH (20 mL) was added KOH(813 mg, 14.5 mmol) in EtOH-H₂O (3 mL:3 mL) dropwise by an additionfunnel at 0° C. over 30 min. The resulting solution was warmed to rt andstirred for 24 h. The mixture was diluted with distilled H₂O (50 mL) andwashed with EtOAc (3×50 mL). The organic layer was dried over Na₂SO₄,filtered and concentrated in vacuo. MeOH-THF (14.5 mL: 14.5 mL) and 12 MHCl (0.7 mL) were added to the residue at 0° C. The resulting solutionwas warmed to rt and stirred for 8 h. The reaction mixture was dilutedwith distilled H₂O (50 mL) and washed with EtOAc (3×50 mL). The organiclayer was dried over Na₂SO₄, filtered and concentrated in vacuo. Theresidue was purified by silica gel chromatography (EtOAc:n-hexane=1:4)to obtain Formula 24a (641 mg, 92%), microcrystalline powder; ¹H-NMR(DMSO-d₆, 300 MHz) δ 12.92 (1H, s), 10.52 (1H, s), 7.59 (2H, s),7.48-7.45 (2H, m), 7.44-7.37 (5H, m), 7.32-7.29 (5H, m), 6.94 (2H, d,J=8.8 Hz), 6.21 (1H, s), 5.16 (2H, s), 5.06 (2H, s), 3.77 (3H, s);¹³C-NMR (DMSO-d₆, 125 MHz) δ 192.0, 160.2, 159.6, 157.7, 153.1, 143.0,136.7, 136.6, 134.4, 130.3, 128.5, 128.4, 128.3, 128.2, 128.0, 127.8,127.4, 124.6, 115.2, 109.0, 99.7, 75.7, 69.4, 60.7; HR-ESI-MS m/z483.1795 [M+H]⁺ (calcd. for C₃₀H₂₇O₆, 483.1802).

Example 8: Preparation of(E)-1-(2-Benzyloxy-4,6-dihydroxy-3-[2H3]methoxyphenyl)-3-(4-benzyloxyphenyl)prop-2-en-1-one(Formula 24b)

According to the procedure as described for Formula 24a, reaction ofFormula 23b (1.0 g, 2.6 mmol), 4-benzyloxybenzaldyde (1.1 g, 5.3 mmol)and KOH (1.5 g, 26.4 mmol) in EtOH (60 mL) followed by treatment of 12 MHCl (1.3 mL) and MeOH-THF (26.4 mL:26.4 mL) gave Formula 24b (1.0 g,81%), yellow microcrystalline powder; ¹H-NMR (DMSO-d₆, 300 MHz) δ 12.95(1H, s), 10.54 (1H, s), 7.59 (2H, s), 7.48-7.45 (2H, m), 7.44-7.36 (5H,m), 7.33-7.29 (5H, m), 6.94 (2H, d, J=8.8 Hz), 6.22 (1H, s), 5.16 (2H,s), 5.06 (2H, s); ¹³C-NMR (DMSO-d₆, 125 MHz) δ 192.0, 160.2, 159.7,157.7, 153.2, 143.0, 136.7, 136.6, 134.4, 130.3, 128.5, 128.4, 128.3,128.2, 128.0, 127.8, 127.4, 124.6, 115.2, 109.0, 99.7, 75.7, 69.4;HR-ESI-MS m/z 486.1983 [M+H]⁺ (calcd. for C₃₀H₂₄D₃O₆, 486.1990).

Example 9: Preparation of4′-Benzyloxy-6-methoxy-5-benzyloxy-7-hydroxyflavone (Formula 25a)

To a solution of Formula 24a (598 mg, 1.2 mmol) in dry DMSO (100 mL) wasadded I₂ (32 mg, 0.1 mmol) in DMSO (3 mL) dropwise by syringe. Theresulting solution was heated to 120° C., and stirred for 2 h. Aftercooling to rt, saturated Na₂S₂O_(3(aq)) (10 mL) was added to thereaction mixture. The mixture was diluted with EtOAc (100 mL) and washedwith distilled H₂O (3×50 mL). The organic layer was dried over Na₂SO₄,filtered and concentrated in vacuo. The residue was purified by silicagel chromatography (EtOAc:n-hexane=1:3) to obtain Formula 25a (555 mg,93%), yellow microcrystalline powder; ¹H-NMR (DMSO-d₆, 300 MHz) δ 10.76(1H, s), 7.98 (2H, d, J=8.9 Hz), 7.61 (2H, d, J=7.0 Hz), 7.48 (2H, d,J=7.0 Hz), 7.44-7.34 (6H, m), 7.18 (2H, d, J=8.9 Hz), 6.91 (1H, s), 6.67(1H, s), 5.22 (2H, s), 5.00 (2H, s), 3.75 (3H, s); ¹³C-NMR (DMSO-d₆, 125MHz) δ 175.8, 160.9, 160.1, 156.1, 153.8, 150.7, 139.6, 137.6, 136.6,128.5, 128.3, 128.1, 128.0, 127.8, 123.4, 115.3, 111.4, 106.0, 100.1,75.6, 69.5, 60.9; HR-ESI-MS m/z 481.1637 [M+H]⁺ (calcd. for C₃₀H₂₅O₆,481.1646).

Example 10: Preparation of4′-Benzyloxy-6-methoxy-5-benzyloxy-7-hydroxyflavone (Formula 25b)

Following the procedure as described for Formula 25a, reaction ofFormula 24b (849 mg, 1.7 mmol) with I₂ (44 mg, 0.2 mmol) in DMSO (120mL) gave Formula 25b (664 mg, 79%), yellow microcrystalline powder;¹H-NMR (DMSO-d₆, 300 MHz) δ 10.75 (1H, s), 7.98 (2H, d, J=8.9 Hz), 7.61(2H, d, J=6.8 Hz), 7.48 (2H, dd, J=1.8, 8.4 Hz), 7.44-7.34 (6H, m), 7.18(2H, d, J=8.9 Hz), 6.91 (1H, s), 6.67 (1H, s), 5.22 (2H, s), 5.01 (2H,s); ¹³C-NMR (DMSO-d₆, 125 MHz) δ 175.8, 160.9, 160.1, 156.1, 153.7,150.7, 139.5, 137.6, 136.6, 128.5, 128.3, 128.1, 128.0, 127.8, 123.4,115.3, 111.4, 106.0, 100.1, 75.6, 69.5; HR-ESI-MS m/z 484.1827 [M+H]⁺(calcd. for C₃₀H₂₂D₃O₆, 484.1834).

Example 11: Preparation of Hispidulin

To a solution of Formula 25a (301 mg, 0.6 mmol) in dry CH₂Cl₂ (40 mL)was added 1 M BCl₃ (2.5 mL, 2.5 mmol) in dry CH₂Cl₂ (5 mL) dropwise bysyringe at −78° C. over 20 min. The resulting solution was stirred for 1h. The reaction mixture was diluted with distilled H₂O (50 mL) andwashed with EtOAc (3×50 mL). The organic layer was dried over Na₂SO₄,filtered and concentrated in vacuo. The residue was purified by silicagel chromatography (EtOAc:n-hexane=1:2) to obtain hispidulin (160 mg,85%), yellow microcrystalline powder; ¹H-NMR (DMSO-d₆, 500 MHz) δ 13.07(1H, s, 5-OH), 10.73 (1H, s, 7-OH), 10.38 (1H, s, 4′-OH), 7.92 (2H, d,J=8.9 Hz, H-2′, H-6′), 6.92 (2H, d, J=8.9 Hz, H-3′, H-5′), 6.77 (1H, s,H-3), 6.59 (1H, s, H-8), 3.74 (3H, s, 6-OMe); ¹³C-NMR (DMSO-d₆, 125 MHz)δ 182.2 (C-4), 163.9 (C-2), 161.2 (C-4′), 157.3 (C-7), 152.8 (C-5),152.4 (C-9), 131.4 (C-6), 128.5 (C-2′, C-6′), 121.2 (C-1), 116.0 (C-3′,C-5′), 104.1 (C-10), 102.4 (C-3), 94.3 (C-8), 60.0 (6-OMe); HR-ESI-MSm/z 301.0702 [M+H]⁺ (calcd. for C₁₆H₁₃O₆, 301.0707). The chemicalstructure of hispidulin was identified by 2D-NMR analyses. FIG. 4 showsthe correlation in the ROESY spectrum of hispidulin that 5-OH (δH 13.07)was correlated to 6-OMe′″ (δH 3.74) and H-8 (δH 6.59) was correlated toH-2′ and H-6′ (δH 7.92). Additionally, the HMBC spectrum showed that5-OH (δH 13.07) correlated to C-5 (δC 152.8), C-6 (δC 131.4), C-7 (δC157.3), C-9 (δC 152.4) and C-10 (δC 104.1); H-8 (δH 13.07) correlated toC-6 (δC 131.4), C-7 (δC 157.3), C-9 (δC 152.4) and C-10 (δC 104.1); and6-OMe-H (δH 3.74) correlated to C-6 (δC 131.4). The ¹H- and ¹³C-NMR dataof synthesized hispidulin were similar to those of hispidulin previouslyisolated.

Example 12: Preparation of d-Hispidulin

Following the procedure as described for hispidulin, reaction of Formula25b (536 mg, 1.1 mmol) in CH₂Cl₂ (75 mL) with 1 M BCl₃ (4.4 mL, 4.4mmol) in CH₂Cl₂ (8.8 mL) gave d-hispidulin (268 mg, 80%), yellowmicrocrystalline powder; ¹H-NMR (DMSO-d₆, 500 MHz) δ 13.07 (1H, s,5-OH), 10.70 (1H, s, 7-OH), 10.36 (1H, s, 4′-OH), 7.91 (2H, d, J=8.9 Hz,H-2′, H-6′), 6.92 (2H, d, J=8.9 Hz, H-3′, H-5′), 6.76 (1H, s, H-3), 6.58(1H, s, H-8); ¹³C-NMR (DMSO-d₆, 125 MHz) δ 182.1 (C-4), 163.8 (C-2),161.2 (C-4′), 157.2 (C-7), 152.8 (C-5), 152.4 (C-9), 131.3 (C-6), 128.5(C-2′, C-6′), 121.2 (C-1), 116.0 (C-3′, C-5′), 104.1 (C-10), 102.4(C-3), 94.2 (C-8); HR-ESI-MS m/z 304.0888 [M+H]⁺ (calcd. for C₁₆H₁₀D₃O₆,304.0895).

Due to the absence of a proton signal of the CD₃O group in the ¹H-NMRspectra of the d-containing intermediate compounds 23b, 24b, 25b andd-hispidulin, these compound structures were identified depending on the¹³C-NMR spectra without ¹H decoupling and the mass technique. The¹³C-NMR spectra revealed a characteristic multiplet splitting pattern ofthe ¹³C signal for the CD₃O group in compounds 23b, 24b, 25b andd-hispidulin. The mass spectra also supported chemical structures ofthese d-labeled compounds. All synthesized compounds had an estimatedpurity of at least 98% as determined by HPLC analysis

Example 13: Human Liver Microsome Stability

Metabolic stability is associated with susceptibility of compounds tobiotransformation. Metabolic half-life (t_(1/2)) and intrinsic clearance(CL_(int)) were compared between hispidulin and d-hispidulin by testingthese synthesized compounds in a human liver microsome stability assay.

Mixed-gender human liver microsomes (Lot #1210347) were purchased fromXenoTech. The reaction mixture minus NADPH was prepared as describedbelow. The test compounds were added into the reaction mixture at afinal concentration of 1 μM. A separate reaction with the controlcompound, testosterone, was conducted simultaneously with the reactionswith the test compounds. An aliquot of the reaction mixture (withoutcofactor) was equilibrated in a shaking water bath at 37° C. for 3 min.After addition of cofactor to initiate the reaction, the mixture wasincubated in a shaking water bath at 37° C. Aliquots (100 μL) werewithdrawn at 0, 10, 20, 30 and 60 min for the test compounds andtestosterone. The reaction was terminated by immediately combining thetested compounds and testosterone samples with 400 μL of ice-cold 50/50acetonitrile (ACN)/H₂O containing 0.1% formic acid and internalstandard. The samples were then mixed and centrifuged to precipitateproteins. All samples were assayed by LC-MS/MS using electrosprayionization. The peak area response ratio (PARR) to internal standard wascompared to the PARR at time 0 to determine the percent remaining ateach time point. The values for half-life (t_(1/2)) and intrinsicclearance (CL_(int)) of the tested compounds were determined byAbsorption System Corp. Half-life calculated using GraphPad software wasfitted to a single-phase exponential decay equation.

The FDA-approved deuterated agent, deutetrabenazine, had a t_(1/2) (8.6h) superior to tetrabenazine (4.8 h). In addition to t_(1/2), the areaunder the curve (AUC) of deutetrabenazine (542 ng·hr/mg) was also higherthan that of its counterpart compound (261 ng·hr/mg) [18]. In thepresent disclosure, hispidulin and d-hispidulin had no significantdifference in t_(1/2) and CL_(int) (Table 3), which suggested that theC6-OMe of hispidulin is resistant to be modified by the human livermicrosome.

TABLE 3 Human liver microsome stability of hispidulin and d-hispidulin.Half-Life Compound (min) CL_(int) ¹ (mL/min/mg Protein) Hispidulin 460.0298 d-Hispidulin 43 0.0325 Testosterone 19 0.0727 ¹Intrinsicclearance (CL_(int)) was calculated based on CL_(int) = k/P, where k isthe elimination rate constant and P is the protein concentration in theincubation.

The references listed below cited in the application are eachincorporated by reference as if they were incorporated individually.

REFERENCES

-   1. Pietta, P. G. Flavonoids as antioxidants. J. Nat. Prod. 2000, 63,    1035-1042.-   2. Serafini, M.; Peluso, I.; Raguzzini, A. Flavonoids as    anti-inflammatory agents. Proc. Nutr. Soc. 2010, 69, 273-278.-   3. Cushnie, T. P.; Lamb, A. J. Antimicrobial activity of flavonoids.    Int. J. Antimicrob. Agents 2005, 26, 343-356.-   4. Cardenas-Rodriguez, N.; Gonzalez-Trujano, M. E.;    Aguirre-Hernandez, E.; Ruiz-Garcia, M.; Sampieri, A., III;    Coballase-Urrutia, E.; Carmona-Aparicio, L. Anticonvulsant and    antioxidant effects of Tilia americana var. mexicana and flavonoids    constituents in the pentylenetetrazole-induced seizures. Oxid. Med.    Cell. Longev. 2014, 2014, 329172.-   5. Guan, L. P.; Liu, B. Y. Antidepressant-like effects and    mechanisms of flavonoids and related analogues. Eur. J. Med. Chem.    2016, 121, 47-57.-   6. Ravishankar, D.; Rajora, A. K.; Greco, F.; Osborn, H. M.    Flavonoids as prospective compounds for anti-cancer therapy. Int. J.    Biochem. Cell Biol. 2013, 45, 2821-2831.-   7. Fan, P. C.; Huang, W. J.; Chiou, L. C. Intractable chronic motor    tics dramatically respond to Clerodendrum inerme (L) Gaertn. J.    Child Neurol. 2009, 24, 887-890.-   8. Huang, W. J.; Lee, H. J.; Chen, H. L.; Fan, P. C.; Ku, Y. L.;    Chiou, L. C. Hispidulin, a constituent of Clerodendrum inerme that    remitted motor tics, alleviated methamphetamine-induced    hyperlocomotion without motor impairment in mice. J. Ethnopharmacol.    2015, 166, 18-22.-   9. Liao, Y. H.; Lee, H. J.; Huang, W. J.; Fan, P. C.; Chiou, L. C.    Hispidulin alleviated methamphetamine-induced hyperlocomotion by    acting at alpha6 subunit-containing GABAA receptors in the    cerebellum. Psychopharmacology 2016, 233, 3187-3199.-   10. Kavvadias, D.; Sand, P.; Youdim, K. A.; Qaiser, M. Z.;    Rice-Evans, C.; Baur, R.; Sigel, E.; Rausch, W. D.; Riederer, P.;    Schreier, P. The flavone hispidulin, a benzodiazepine receptor    ligand with positive allosteric properties, traverses the    blood-brain barrier and exhibits anticonvulsive effects. Br. J.    Pharmacol. 2004, 142, 811-820.-   11. Shi, Z. H.; Li, N. G.; Wang, Z. J.; Tang, Y. P.; Dong, Z. X.;    Zhang, W.; Zhang, P. X.; Gu, T.; Wu, W. Y.; Yang, J. P. et. al.    Synthesis and biological evaluation of methylated scutellarein    analogs based on metabolic mechanism of scutellarin in vivo. Eur. J.    Med. Chem. 2015, 106, 95-105.-   12. Lin, H.; Zhang, W.; Dong, Z. X.; Gu, T.; Li, N. G.; Shi, Z. H.;    Kai, J.; Qu, C.; Shang, G. X.; Tang, Y. P. et. al. A new and    practical synthetic method for the synthesis of    6-O-methyl-scutellarein: One metabolite of scutellarin in vivo.    Int. J. Mol. Sci. 2015, 16, 7587-7594.-   13. Chao, S. W.; Su, M. Y.; Chiou, L. C.; Chen, L. C.; Chang, C. I.;    Huang, W. J. Total synthesis of hispidulin and the structural basis    for its inhibition of proto-oncogene kinase Pim-1. J. Nat. Prod.    2015, 78, 1969-1976.-   14. Shen, M. Z.; Shi, Z. H.; Li, N. G.; Tang, H.; Shi, Q. P.;    Tang, Y. P.; Yang, J. P.; Duan, J. A. Efficient Synthesis of    6-O-methyl-scutellarein from Scutellarin via selective methylation.    Lett. Org. Chem. 2013, 10, 733-737.-   15. Zhang, W.; Dong, Z. X.; Gu, T.; Li, N. G.; Zhang, P. X.; Wu, W.    Y.; Yu, S. P.; Tang, Y. P.; Yang, J. P.; Shi, Z. H. A new and    efficient synthesis of 6-O-methylscutellarein, the major metabolite    of the natural medicine scutellarin. Molecules 2015, 20,    10184-10191.-   16. Katsnelson, A. Heavy drugs draw heavy interest from pharma    backers. Nat. Med. 2013, 19, 656.-   17. Gant, T. G. Using deuterium in drug discovery: leaving the label    in the drug. J. Med. Chem. 2014, 57, 3595-3611.-   18. Tung, R. D. Deuterium medicinal chemistry comes of age. Future    Med. Chem. 2016, 8, 491-494.

1. A method for preparing hispidulin or a derivative thereof fromtrihydroxybenzaldehyde, the method comprising: providing an intermediatecompound represented by following Formula (IV):

performing alkylation and Claisen-Schmidt condensation of theintermediate compound, followed by deprotection to obtain a compoundrepresented by following Formula (IA):

performing cyclization of the compound represented by Formula (IA) inthe presence of a catalyst to obtain a compound represented by followingFormula (IB):

deprotecting the compound represented by Formula (IB) to obtain thehispidulin or the derivative thereof having following formula:

wherein PG₁, PG₂, PG₃ and PG₄ are each independently a hydroxylprotecting group selected from the group consisting of methoxymethyl(MOM), 2-methoxyethoxymethyl (MEM), ethoxymethyl (EOM), t-butoxymethyl,benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), allyloxymethoxy,tetrahydropyranyl (THP), methylthiomethyl (MTM),tri-1-propylsilyloxymethyl (TOM), (phenyldimethylsilyl)methoxymethyl(SMOM), acetyl, pivaloyl (Piv), benzoate, methyl, ethyl, benzyl (Bn),p-methoxybenzyl (PMB), triphenylmethyl (Tr), methoxytrityl (MMT),dimethoxytrityl (DMT), trimethylsilyl (TMS), t-butyldimethylsilyl(TBDMS), triisopropylsilyl (TIPS), tri(trimethylsilyl)silyl (TTMSS), andt-butyldiphenylsilyl (TBDPS), and R is hydrogen, an optionallysubstituted alkyl or an optionally substituted cycloalkyl.
 2. (canceled)3. The method of claim 1, wherein the trihydroxybenzaldehyde is2,4,6-trihydroxybenzaldehyde.
 4. (canceled)
 5. The method of claim 1,wherein the optionally substituted alkyl is selected from the groupconsisting of CH₃, C₂H₅, and C₃H₇.
 6. The method of claim 1, wherein theoptionally substituted cycloalkyl is C₆H₅CH₂.
 7. The method of claim 1,wherein the hispidulin derivative is deuterium-labeled.
 8. The method ofclaim 7, wherein the deuterium-labeled hispidulin derivative hasfollowing formula:

wherein R is CD₃.
 9. The method of claim 1, wherein the intermediatecompound is obtained by Baeyer-Villiger oxidation and basic hydrolysisof a compound represented by following Formula (V):

wherein PG₁, PG₂ and PG₃ are as defined in claim
 1. 10. The method ofclaim 1, wherein the trihydroxybenzaldehyde reacts with at least oneprotecting group to obtain a compound represented by following Formula(VII):

wherein PG₂ and PG₃ are as defined in claim
 1. 11. The method of claim10, wherein the compound represented by Formula (VII) undergoesregioselective iodination and reacts with an additional protecting groupto obtain a compound represented by following Formula (VI):

wherein PG₁, PG₂ and PG₃ are as defined in claim
 1. 12. The method ofclaim 11, further comprising conducting Stille coupling to obtain thecompound represented by following Formula (V):

wherein PG₁, PG₂ and PG₃ are as defined in claim
 1. 13. The method ofclaim 12, wherein the Stille coupling is conducted with a palladiumcatalyst and an organic solvent.
 14. The method of claim 13, wherein thepalladium catalyst is Pd(PPh₃)₄, PdCl₂(PPh₃)₂, Pd(dppf)Cl₂, PdCl₂,Pd(OAc)₂, Pd(dba)₂, PdCl₂(MeCN)₂, BnPdCl(PPh₃)₂, or C₄H₆Br₂N₂Pd.
 15. Themethod of claim 13, wherein the organic solvent is toluene, dioxane,tetrahydrofuran, acetonitrile, chloroform, dichloromethane,chlorobenzene, dimethylacetamide, methylpyrrolidone, dimethyl sulfoxide,or hexamethylphosphoramide. 16-17. (canceled)
 18. The method of claim 1,wherein the catalyst in the cyclization is catalytic 12, potassiumiodide, ammonium iodide, tetra-(n-butyl)ammonium iodide, selenium (IV)oxide, dihydrogen peroxide, cerium (IV) sulfate tetrahydrate,2,3-dicyano-5,6-dichloro-p-benzoquinone or bis(acetoxy)iodobenzene. 19.(canceled)
 20. The method of claim 1, wherein the deprotecting isdebenzylation of the compound represented by Formula (IB) in a reactionwith BCl₃, hydrogen, palladium on activated carbon, titaniumtetrachloride, boron tribromide, acetic acid or methanesulfonic acid.